WO2020135713A1 - Procédé et produit pour détecter l'état de santé d'un embryon à l'aide d'une solution de culture de blastocyste - Google Patents

Procédé et produit pour détecter l'état de santé d'un embryon à l'aide d'une solution de culture de blastocyste Download PDF

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WO2020135713A1
WO2020135713A1 PCT/CN2019/129196 CN2019129196W WO2020135713A1 WO 2020135713 A1 WO2020135713 A1 WO 2020135713A1 CN 2019129196 W CN2019129196 W CN 2019129196W WO 2020135713 A1 WO2020135713 A1 WO 2020135713A1
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blastocyst
culture
cultured
module
embryo
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PCT/CN2019/129196
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Chinese (zh)
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姚雅馨
李文璐
马杰良
陆思嘉
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序康医疗科技（苏州）有限公司
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Priority to JP2021538406A priority Critical patent/JP2022516543A/ja
Priority to CN201980068400.7A priority patent/CN113286892B/zh
Priority to US17/418,934 priority patent/US20220112549A1/en
Priority to EP19903766.4A priority patent/EP3904529A1/fr
Publication of WO2020135713A1 publication Critical patent/WO2020135713A1/fr

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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/6858—Allele-specific amplification
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    • C12M41/00—Means for regulation, monitoring, measurement or control, e.g. flow regulation
    • C12M41/46—Means for regulation, monitoring, measurement or control, e.g. flow regulation of cellular or enzymatic activity or functionality, e.g. cell viability
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0603—Embryonic cells ; Embryoid bodies
    • C12N5/0604—Whole embryos; Culture medium therefor
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/6848—Nucleic acid amplification reactions characterised by the means for preventing contamination or increasing the specificity or sensitivity of an amplification reaction
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material

Definitions

  • the present invention relates to the fields of biomedicine and molecular cell biology, and in particular, to a method and product for detecting embryo health status using blastocyst culture fluid.
  • PES Preimplantation Genetic Screen detects the chromosome status of embryos cultured in vitro to screen for embryos with normal chromosomes to be placed in the mother's womb, thereby increasing the success rate of conception to about 60%.
  • the biological samples required for various tests are one to several cells collected from embryos cultured in vitro, commonly known as biopsies. The detection of these few cells reflects whether the chromosomes of the entire embryo are normal. Theoretically, the chromosomal state of several cells taken out is the same as that of other cells in the embryo. Examining these cells can know whether the chromosomal state of the embryo is normal. It is generally believed that the absorption of several trophoblast cells at this time will not adversely affect embryonic development.
  • the specific conventional treatment method is: fresh in vitro fertilized eggs are cultured in the culture medium to D3, and the embryos are transferred to the blastocyst culture medium at D3 to continue to culture to D5 or D6, and the blastocysts are removed from D5 or D6 to observe the embryo rating. After the freezing level is reached, the freezing operation is carried out immediately, and the remaining blastocyst culture fluid is the sample to be collected during the test.
  • In vitro fertilization methods are usually selected in IVF and ICSI. IVF is accomplished by co-cultivation of multiple sperm and oocytes, and ICSI is completed by single sperm injection. IVF embryos usually have extra sperm hanging around the zona pellucida. In order to avoid parental DNA interference of sperm, ICSI fertilization is generally adopted in the above conventional processing methods.
  • this method uses D3 to replace the culture medium, and the embryos in the blastocyst culture medium Culture from D3 to D5/D6, and then collect blastocyst culture fluid as the test sample.
  • Vera-Rodriguez M., etc. Origin and composition of cell-free DNA in spent medium from human culture culture during training plant development, Hum Reprod.
  • this document also uses D3 to replace culture
  • this blastocyst culture solution embryos are cultured from D3 to D5/D6, and then blastocyst culture solution is collected as a test sample, and only for ICSI fertilization.
  • CN201510746098.X is also directly cultured to D5/D6 after changing culture medium from D3, and is only for ICSI fertilization.
  • the accuracy of embryo results is greatly improved, but there is still a false negative rate of about 15%, mainly because the test sample is inevitably subject to external interference, especially maternal interference.
  • in vitro operations such as transplantation or freezing can be considered after the blastocysts are encapsulated, so clinically, in vitro operations such as changing the culture medium of embryos during the mulberry stage of D4 are not usually performed, so as not to affect the cyst formation rate.
  • the current detection method is inevitably subject to external interference, especially maternal interference, resulting in a false negative rate that is too high to target both ICSI and IVF samples.
  • the purpose of the present invention is to provide a method and a product suitable for ICSI and IVF that can improve the accuracy and reduce the false negative rate by using the blastocyst culture fluid as the detection sample, thereby detecting the health status of the embryo.
  • the first aspect of the present invention provides a method of using blastocyst culture fluid to detect the blastocyst health status in vitro, which includes the steps of:
  • the first blastocyst culture system includes a de novo cultured blastocyst culture system (that is, culture is started from D1 without changing the medium).
  • the first blastocyst culture system includes the last blastocyst culture system after one or more fluid exchanges (for example, the fluid exchange culture is performed on D2-D3 or D3-D4, for example At D3).
  • "cultivation days are D5-D6" includes D5 ⁇ 12 hours or D6 ⁇ 12 hours, preferably D5 ⁇ 6 hours, more preferably D5 to D5+6 hours.
  • the number of culture days is D5, that is, the 5th day after the start of the culture, and the start of the culture is set to the first day (D1).
  • step (a) the blastocyst culture in vitro is de novo culture (that is, culture is started from D1, and the medium is not changed).
  • step (a) the blastocyst culture in vitro is staged culture.
  • the blastocyst in vitro culture includes first-stage blastocyst culture and second-stage blastocyst culture.
  • the first stage blastocyst culture includes D1 to D3, preferably, D1 to D3 ⁇ 12h, more preferably, D1 to D3 ⁇ 8h, more preferably, D1 to D3 ⁇ 6h , Cultivate with the culture medium of the cleavage stage.
  • the second-stage blastocyst culture includes D3 to D5, and is cultured with blastocyst culture fluid.
  • the T1 time is 2-8h, such as 3-6h, preferably 3-5h.
  • the zona pellucida is perforated in the second blastocyst culture system.
  • the hole diameter of the transparent tape is 10-40 ⁇ m, preferably 10-30 ⁇ m, more preferably 10-20 ⁇ m.
  • the method has one or more of the following characteristics:
  • the signal-to-noise ratio is high, the signal-to-noise ratio S 1 /S 0 >2, preferably, >5, more preferably, >10, where S 1 is the signal from the embryo and S 0 is the signal from the background;
  • the first blastocyst culture system is a single-sperm fertilization culture system (or ICSI culture system).
  • the first blastocyst culture system is a multi-sperm fertilization culture system (or IVF culture system).
  • the blastocyst cultured in vitro is a single-sperm fertilized blastocyst (or ICSI blastocyst).
  • the blastocyst cultured in vitro is a polysperm fertilized blastocyst (or IVF blastocyst).
  • the second blastocyst culture system contains only one blastocyst (embryo).
  • the second blastocyst culture system is a single embryo culture system, and the single embryo culture system contains 10-60 microliters, preferably, 10-50 microliters, or 10- 30 microliters, more preferably, 10-15 microliters of culture medium.
  • the volume of the culture medium taken out is 50-100% of the volume of the culture medium in the single embryo culture system, preferably, 70-100%, more preferably , 80-100%, optimally, 90-100%.
  • step (d) further includes step (e):
  • the health status of the embryo includes: chromosome aneuploidy, mitochondrial copy number, whether the DNA content is normal, and detection of pathogenic genes.
  • the genome analysis method includes NICS-INST amplification method and analysis method.
  • NICS-INST amplification method of NICS-INST
  • amplification method of NICS-INST please refer to the Gene Sequencing Generic Library Kit (trade name NICS-Inst TM , Generic Name: Gene Sequencing Generic Library Kit; English name: Universal Library Preparation Kit) ).
  • Other genome analysis methods known in the art, including amplification methods and analysis methods, are also applicable to the present invention.
  • CN103890191B discloses using MALBAC genome amplification method and the like.
  • the method of genomic analysis is selected from the group consisting of second generation sequencing, nucleic acid chip, immunofluorescence detection, fluorescent PCR detection, first generation sequencing, third generation sequencing, mass spectrometry detection, or a combination thereof.
  • the lysate contains an ingredient selected from the group consisting of Tris buffer, chelating agent, hydrochloride, nonionic surfactant, or a combination thereof.
  • the Tris buffer includes Tris-Cl.
  • the concentration of the Tris buffer is 10-60 mM, preferably 15-50 mM, more preferably 20-45 mM, such as 30 mM.
  • the pH of the Tris buffer is 5-10, preferably, 6-9, and more preferably, 7-8.
  • the chelating agent includes EDTA.
  • the concentration of the chelating agent is 0.2-8 mM, preferably, 0.3-6 mM, more preferably, 0.5-4 mM, such as 2 mM.
  • the hydrochloride salt is selected from the group consisting of KCl, NaCl, or a combination thereof.
  • the concentration of the hydrochloride salt is 5-60 mM, preferably, 8-40 nM, more preferably, 10-40 mM, such as 20 mM.
  • the nonionic surfactant is selected from the group consisting of Triton X-100, Triton X-114, Tween 20, NP40, SDS, or a combination thereof.
  • the concentration of the nonionic surfactant is 0.02-10%, preferably, 0.05-5%, more preferably, 0.1-3%, for example, 0.2%, with the cleavage The total weight of the liquid.
  • step (e) (i) the volume ratio of the culture solution to the lysate is 1:10-10:1, preferably, 1:5-5:1, more preferably Ground, 1:2-2:1.
  • the lyase is selected from the group consisting of proteinase K, Qiagen Protease, pepsin, papain, trypsin, lysozyme, or a combination thereof.
  • the concentration of the lyase is 1-25 ⁇ g/ml, preferably, 5-20 ⁇ g/ml, and more preferably, 10-15 ⁇ g/ml.
  • the added amount of the lyase is 0.1-10 ⁇ l, preferably, 0.5-6 ⁇ l, and more preferably, 0.8-3 ⁇ l.
  • step (e)(ii) includes one or more features selected from the group consisting of:
  • the incubation temperature is 20-70°C, preferably, 30-60°C;
  • the incubation time is 1min-12h, preferably, 10min-6h, more preferably, 30min-2h;
  • the deactivation temperature is 60-100°C, preferably 75-95°C;
  • the inactivation time is 0.5-20 min, preferably 0.8-15 min.
  • genomic analysis is performed by PCR, preferably the PCR reaction tube contains an amplification mixture, 0.5%-20% PCR inhibitor antagonist, 5-20mM dNTP, 5-100 ⁇ M NG and NT primers, 50-200 ⁇ M amplification primers, 0.5-10 unit nucleic acid polymerase, preferably, the PCR inhibitor antagonist is selected from DMSO, betaine, formamide, glycerol and albumin
  • the nucleic acid polymerases are selected from Phi29 DNA polymerase, Bst DNA polymerase, Vent polymerase, Deep Vent polymerase, Klenow Fragment DNA polymerase I, MMLV reverse transcriptase, AMV reverse transcriptase , HIV reverse transcriptase, ultra-fidelity DNA polymerase, Taq polymerase, E. coli DNA polymerase, LongAmp Taq DNA polymerase and OneTaq DNA polymerase one or more.
  • the components of the amplification mixture are 10-25mM Tris-HCl, 5-25mM (NH4) 2SO4, 5-30mM KCl, 0.5-5mM MgSO4, 0.1%-20% DMSO and 0.05- 5% Triton X-100.
  • the components of the amplification mixture are preferably 15 mM Tris-HCl, 15 mM (NH4) 2SO4, 20 mM KCl, 1 mM MgSO4, 5% DMSO, and 2% Triton X-100.
  • the NG and NT primers contain a universal sequence and a variable sequence from the 5'end to the 3'end, wherein the universal sequence is composed of three of the four bases of G, A, C and T Or two components, provided that the universal sequence does not include G and C at the same time; the amplification primer contains the universal sequence and does not contain the variable sequence.
  • variable sequence is selected from the group consisting of: (N)nGGG, (N)nTTT, (N)mTNTNG, (N)xGTGG(N)y, where N is any natural nucleic acid
  • N is any natural nucleic acid
  • n is a positive integer selected from 3-17
  • m is a positive integer selected from 3-15
  • x and y are positive integers selected from 3-13, respectively.
  • step (e)(iii) the thermal cycling procedure of whole genome amplification when performing genome analysis by PCR is as follows:
  • step (e)(iii) the thermal cycling procedure of whole genome amplification when performing genome analysis by PCR is as follows:
  • the annealing temperature is 50 °C for 45s;
  • the method is non-therapeutic and non-diagnostic.
  • the second aspect of the present invention provides a method for preparing a genetic test sample or a chromosome test sample, including the steps of:
  • the cultured blastocyst culture solution is taken out to obtain a cell-free cultured blastocyst culture solution, which is a test sample.
  • the second blastocyst culture system contains only one blastocyst (embryo).
  • the second blastocyst culture system is a single embryo culture system, containing 10-60 microliters, preferably, 10-50 microliters, more preferably, 10-15 microliters of culture medium .
  • the volume of the detection sample is 50-100% of the volume of the culture solution in the second blastocyst culture system, preferably, 70-100%, more preferably , 80-100%, optimally, 90-100%.
  • a third aspect of the present invention provides a kit for detecting blastocyst health using blastocyst culture fluid, including:
  • the first container, the second container, and the third container are different.
  • the cleavage stage culture fluid in the first container is used for D1 to D3, preferably, D3 ⁇ 12h, more preferably, D3 ⁇ 8h, more preferably, D3 ⁇ 6h, Cleavage embryo culture.
  • the first blastocyst culture solution in the second container is used for blastocyst culture of D3-D5.
  • the second blastocyst culture solution in the third container is used for blastocyst culture after D5-D6.
  • the components of the first blastocyst culture fluid and the second blastocyst culture fluid are the same, both of which are G-2 PLUS medium (Vitrolife) or Quinn's Advantage Protein Plus blastocyst medium (SAGE) blastocyst culture fluid .
  • the cleavage stage culture fluid is G-1 PLUS medium (Vitrolife) or Quinn’s Embryo maintenance medium (SAGE) cleavage stage culture fluid.
  • a fourth aspect of the present invention provides a device for assisting in diagnosing the health status of blastocysts, including:
  • a culture module the culture module includes a blastocyst late culture module, the blastocyst late culture module is used to change the medium of D5-D6 cultured blastocysts, and to perform blastocyst late culture for a period of time (T1 );
  • sampling module is used to take out the culture fluid of the blastocyst cultured in the later stage of the blastocyst culture module, and used as a test sample;
  • the detection module is used for genetic testing of the detection sample from the sampling module to obtain the detection result
  • the judgment processing module of the health status of the blastocyst evaluates the health status of the embryo to obtain an evaluation result of the health status of the blastocyst
  • the T1 time is 2-8h, such as 3-6h, preferably 3-5h.
  • FIG. 1 shows a schematic diagram of a conventional sampling method different from the sampling method of the present invention in detecting the exposure time of a sample.
  • Figure 2 shows the results of CNV analysis of the culture fluid collected using the conventional D3-D5 sampling method.
  • Fig. 3 shows the results of CNV analysis of the culture fluid collected on the same day using the D5 sampling method of the present invention.
  • the inventors unexpectedly discovered for the first time that in the 10-20 microliter blastocyst culture system of the present invention, the embryo (blastocyst) is placed in the blastocyst culture solution After culturing to D5-D6 days, the embryos were transferred to fresh blastocyst culture medium for cultivation, and a small amount of culture medium was removed from the fresh blastocyst culture medium for testing.
  • the health status of the obtained embryos (such as (Chromosome aneuploidy, mitochondrial copy number, whether the DNA content is normal, pathogenic gene detection)
  • the detection result has extremely high accuracy, and the false negative rate is extremely low ( ⁇ 8%, preferably, ⁇ 5%) , Can greatly eliminate the risk of contamination and interference of excess sperm and maternal granulosa cells.
  • the inventor has completed the present invention.
  • blastocyst and “embryo” are used interchangeably and refer to the final stage of embryo culture in vitro, which usually forms on the 5th-7th day after egg fertilization.
  • depleted medium refers to a culture medium that has been used, for example, a culture medium separated from a culture system in which embryos have been cultured.
  • de novo culture refers to an embryo culture process in which the culture medium is not replaced from the beginning of the culture process to the end of the culture process.
  • the de novo culture of D1-D5 means that the embryo culture process starts from D1 and continues to D5, and the culture medium is not replaced during this period.
  • staged culture refers to an embryo culture process in which embryos undergo one or more fluid exchanges.
  • D1-D5 culture in stages means that the embryo culture process starts from D1 and continues to D5, but during the embryo undergoes one or more fluid changes.
  • D1-D3 embryos are cultured in the culture medium of the cleavage stage; while in D3-D5, embryos are cultured in blastocyst culture medium.
  • D3-D5 sampling means that one culture broth is used to culture embryos on days D3 to D5, and a sample is collected from the used culture broth (ie, spent culture broth) at D5. Therefore, the culture fluid sample obtained in this sampling manner will have a contact time with the embryo culture (ie, the exposure time of the test sample) of at least about 48 hours or more.
  • “same day” sampling means that, on the day when the culture medium is changed, after a short period of embryo culture, a sample is collected from the used culture medium (ie, the spent culture medium). Therefore, the culture fluid sample obtained in this sampling manner will have a short contact time with the embryo culture (ie, the exposure time of the test sample). In the present invention, it is preferred that the exposure time does not exceed 8 hours, such as 3-6 hours, and more preferably 3-5 hours.
  • lysate refers to a lysis buffer used to decompose proteins and cells in the culture fluid sample.
  • the lysate may or may not contain lyase.
  • the lysate and lyase may be added to the culture fluid sample at the same time. In other embodiments, the lyase is added after the lysate is mixed with the culture fluid sample.
  • test sample in vitro incubation time means that the culture medium taken from the sample has already contacted the embryo in vitro (ie, incubated with the embryo ) Length of time.
  • CNV refers to chromosome copy number variation (Copy number variants). CNV naming adheres to the International Human Cell Genome Naming System (ISCN). See, Shaffer LG, Slovak ML, Campbell LJ (2009): ISCN 2009 international system for human cytogenetic nomenclature, Human Genetics volume 126, Article number: 603 (2009).
  • IVF in vitro fertilization
  • in vitro fertilization in vitro fertilization, in vitro fertilization
  • IVF embryo transfer technology
  • IVF embryo transfer technology
  • Multi-sperm fertilized blastocyst or “IVF blastocyst” refers to a blastocyst obtained by using IVF technology to complete fertilization by co-cultivating multiple sperm and oocytes in vitro.
  • ICSI Intracytoplasmic Sperm Injection
  • Single sperm fertilized blastocyst or “ICSI blastocyst” refers to a blastocyst obtained by using ICSI in the cytoplasm of sperm in the egg.
  • the present invention provides a detection method for genetic testing of depleted medium (that is, culture fluid separated from a culture system where blastocysts have been cultured) after blastocyst culture, thereby identifying The health of the embryo.
  • depleted medium that is, culture fluid separated from a culture system where blastocysts have been cultured
  • the method of performing genetic detection on the "deficient" culture medium after blastocyst culture is not particularly limited, and conventional methods can be used for detection, such as second-generation sequencing, nucleic acid chip, immunofluorescence detection, fluorescent PCR detection, One-generation sequencing, three-generation sequencing, mass spectrometry, or a combination thereof.
  • the detection method includes the following steps:
  • step (d) further includes step (e):
  • the main steps of the method for using the blastocyst culture medium as a test sample to reflect the health status of the embryo include:
  • Oocytes are fertilized by IVF or ICSI. After fertilization, the fertilized eggs are transferred to the culture medium to the D3 cleavage stage, and the embryos are transferred to the blastocyst culture medium at D3 and cultured to D5-D6;
  • step (c) Transfer the original blastocyst culture solution obtained in step (b) to the lysate. After centrifugation, the sample enters the next step of the whole genome amplification step, including but not limited to the NICS-INST amplification method and analysis method To get the test result reflecting the health of the embryo.
  • the invention replaces the culture medium on D5 ⁇ 0.5 days, and after a short period of 3-5 hours of culture, the blastocyst culture medium is obtained as the test sample, the accuracy of the test result is significantly improved, the false negative rate is reduced to less than 5%, and the external source is avoided to the greatest extent. Interference is especially from maternal interference, and both ICSI and IVF fertilization methods are applicable.
  • the principle that the accuracy of the test result is significantly improved is that: first, the exposure time of the test sample is extremely short, and the exposure time of 2-3 days is greatly shortened compared to the conventional operation mode (as shown in FIG. 1), and the maximum avoidance External interference, especially maternal interference, is not restricted by the method of fertilization, which greatly improves the accuracy of the test results.
  • the abnormal fragments will be released into the medium during the cultivation in the conventional manner, which may have an impact on the accuracy; and the sampling of the D5 of the present invention after 3-5 hours of culture replacement makes the embryo in the replacement
  • the culture time in the culture medium is short, and the embryo has completed self-repair in the D3-D5 culture stage before D5 is changed to the culture medium.
  • the abnormal fragments are mainly released in the D3-D5 stage, so the abnormal fragments are released in the detection samples collected by the present invention. The probability is low, so the accuracy of the detection results is improved.
  • no one tried to collect culture fluid of IVF embryos but generally collected culture fluid of embryos produced by ICSI fertilization.
  • the most important factor for exogenous interference is that the detection sample is incubated for too long in vitro.
  • the present invention adopts a short-term incubation medium collection method to avoid external interference such as sperm and granule cells due to too long incubation time. These interferences will cause embryonic The decrease in the specific gravity of DNA affects the accuracy of the results.
  • the amount of the test sample in step (c) is 8-15ul, preferably 10ul.
  • the amount of the new blastocyst culture fluid ie, the second blastocyst culture fluid
  • the blastocyst rating includes selecting embryos that have developed to stages 4 or above as well-developed embryos.
  • the punching operation in step (b) increases the release of blastocyst cavity fluid, that is, the release of free nucleic acids, thereby satisfying the requirement that the initial amount of nucleic acid substance in the test sample is increased under the premise of 3-5 hours of short-term incubation, which is sufficient Subsequent amplification and detection ensure that the success rate of the detection can reach more than 97%.
  • embryo observation, grading and cryopreservation are carried out in the blastocyst stage. It is believed that the operation in the blastocyst stage will not damage the embryo.
  • the present invention also chooses to perform laser drilling and in vitro cultivation in the D5 blastocyst stage, so it does not affect Under the premise of embryonic development.
  • step (b) may further include a step of washing embryos before D5 is changed to a culture medium. This step is helpful to improve the accuracy of the detection result.
  • step (a) after the blastocyst culture solution is replaced by D3, multiple embryos can be mixed and cultured, or single embryo culture can be performed.
  • the invention also provides a method for preparing a gene detection sample or a chromosome detection sample, which includes the steps of:
  • the cultured blastocyst culture solution is taken out to obtain a cell-free cultured blastocyst culture solution, which is a test sample.
  • test sample obtained by the method can be used for genetic testing to identify the health status of the embryo.
  • a preferred aperture diameter of the transparent belt is 10-40 ⁇ m, preferably, 10-30 ⁇ m, and more preferably, 10-20 ⁇ m.
  • the punching operation of the transparent zone increases the release of blastocyst cavity fluid, that is, the release of free nucleic acid, which satisfies the premise that the initial amount of nucleic acid substance in the test sample increases under the premise of 3-5 hours of short-term incubation , Enough for subsequent amplification and detection, the success rate of detection can reach more than 97%.
  • the success rate of detection is also very high, about 70%.
  • kits for detecting embryo health status using blastocyst culture fluid including:
  • a device for assisting diagnosis of the health status of an embryo including:
  • a culture module includes a blastocyst late culture module, and the blastocyst late culture module is used to change the medium of D5-D6 cultured blastocysts and perform blastocyst late culture for T1 time;
  • sampling module is used to take out the culture solution of the blastocyst after culturing T1 time in the blastocyst late culture module, and use it as a test sample;
  • the detection module is used for genetic testing of the detection sample from the sampling module to obtain the detection result
  • a discrimination processing module for the health status of the embryo evaluates the health status of the embryo based on the detection result, thereby obtaining an evaluation result of the health status of the embryo;
  • the invention also relates to the use of the module (a), optionally in combination with modules (b)-(e), in the preparation of equipment for the method for evaluating the blastocyst health of the invention.
  • the present invention can adopt a single embryo culture system, that is, only one embryo is cultured in one culture solution, and the detection result of the health status of the embryos obtained by the system is more accurate.
  • the method of the present invention is a general method, which can be used for single-sperm fertilized embryos, and can also be used for multi-sperm fertilized embryos, preferably multi-sperm fertilized embryos.
  • the transparent tape can be perforated, and the results obtained will be better.
  • the method of the present invention has a very high signal-to-noise ratio.
  • the method of the present invention collects the embryo culture fluid after the embryo is cultured to D5, the operation time is extremely short, the test sample culture time is controllable, and the possibility of external interference is the lowest, in addition, the sample collection is not limited to fertilization
  • the method is a medium collection method that is suitable for IVF and ICSI embryos.
  • the D5 medium replacement short-term culture program of the present invention has the advantage of controlling the incubation time of the test sample in vitro, thereby maximizing the control of exogenous interference such as maternal interference and sperm interference, making the application of the present invention not Limited to ICSI embryos, it can also be applied to IVF embryos.
  • Fertilized eggs in volunteer couples, eggs are obtained from female volunteers, sperm are obtained from male volunteers, and IVF or ICSI is used for in vitro fertilization, that is, fertilized eggs) are cultured in vitro until the third day, Cultivate to 5th/6th day with or without fluid exchange;
  • step (3) Transfer the culture medium of short-term cultured blastocysts (volume is about 10-15 ⁇ l) obtained in step (2) to 5 ⁇ l of lysate (pH 7.8 30 mM Tris-Cl, 2 mM EDTA, 20 mM KCl , 0.2% Triton X-100), mark the sample name on the collection tube with a marker. Centrifuge for 30 seconds in a microcentrifuge. Samples can be immediately entered into the next step of whole genome amplification or stored frozen at -20°C or -80°C.
  • the detection method of the present invention refers to the instruction manual of Gene Sequencing Generic Library Kit (trade name NICS-Inst TM , Generic Name: Gene Sequencing Generic Library Kit; English name: Universal Library Preparation Kit) of Sukang Medical Technology (Suzhou) Co., Ltd. get on.
  • This embodiment relates to a comparison between the D5 sampling method of the present invention and the conventional D3-D5 sampling method.
  • the fertilized egg is cultured to D3 in vitro, and the resulting embryo is transferred to the blastocyst culture medium, and cultured to D5.
  • the blastocyst culture fluid of D5 is taken as the "D3-D5 sampling” sample.
  • D5 blastocysts are transferred to a new blastocyst culture medium, and on the day of D5, according to the description of the previous general method, the day of D5 is sampled. Compare the CV test result of "D3-D5 Sampling" from the same embryo with the test result of "D5 Sampling on the same day” and the test result of the whole embryo.
  • the test result of the whole embryo is abnormal chromosome copy number, and the test result of the culture medium is normal, the test result of the culture medium is judged as a false negative, and the probability of false negatives as a whole is the false negative rate.
  • the false negative rate of D5 replacement blastocyst culture medium after short-term culture sampling can be controlled at 1.6%; while in the D3-D5 sampling method, the maternal interference cannot be effectively controlled, and the false negative rate is 12.3%, easy to cause clinical misdiagnosis.
  • FIG. Figures 2 and 3 show the chromosome copy number (CN) map of a male embryo using the method of the present invention and the conventional D3-D5 sampling method.
  • the horizontal axis of the CN diagram is the chromosome, which is arranged in the order of autosomes 1-22, sex chromosomes X, Y; the ordinate axis is the copy number.
  • the autosomal copy number should be 2
  • the sex chromosomes are two copies of X (female), or one copy of each of X and Y (male).
  • the culture fluid collected on the day of D5 can minimize the interference of maternal sources and restore the true CNV results, as shown in FIG. 3, where the interpretation of CNV is 46XY (male).
  • the method of the present invention has very good accuracy and a very low false negative rate.
  • the method is the same as that in Example 1.
  • the difference is that the culture is changed to D3 to D4, the medium is changed at D4, and after 24 or 48 hours of continuous culture, the blastocyst culture medium is taken for detection.
  • the test results on 19 groups of samples show that the accuracy of the test results of embryo health (such as embryo chromosome abnormality) is 84.2 (16/19)%, and the false negative rate is 10.5 (2/19)%.
  • the method of Comparative Example 1 was used, and the results showed that the accuracy of the method was smaller and the false negative rate was higher.
  • D4 embryos usually develop into mulberry stage embryos, which is the key period for embryos to develop into blastocysts. The next stage of morula embryos is the blastocyst stage.
  • the rate of cyst formation is a key indicator of embryo development.
  • In vitro operations such as freezing are performed, so clinically, in vitro operations such as fluid exchange of embryos during the mulberry stage of D4 are generally not performed, so as not to affect the cyst formation rate.
  • the present invention chooses to perform laser drilling and in vitro culture at the D5 blastocyst stage, so it is carried out on the premise of not affecting embryo development.

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Abstract

L'invention concerne un procédé et un produit pour détecter l'état de santé d'un embryon au moyen d'une solution de culture de blastocystes. Plus particulièrement, l'invention concerne un procédé de détection de l'état de santé d'un embryon in vitro à l'aide d'une solution de culture de blastocystes, consistant (a) à fournir un premier système de culture de blastocystes contenant un blastocyste étant cultivé pendant cinq à six jours in vitro; (b) à transférer le blastocyste dans un second système de culture de blastocyste contenant une nouvelle solution de culture de blastocyste pour une culture d'échange de solution, la période pour la culture d'échange de solution étant T1, obtenant ainsi une solution de culture de blastocyste cultivée; (c) à prélever la solution de culture de blastocyste cultivée, ce qui permet d'obtenir une solution de culture de blastocyste cultivée exempte de cellules, c'est-à-dire un échantillon de détection; et (d) à effectuer une détection de gène sur l'échantillon de détection, ce qui permet de déterminer l'état de santé dudit blastocyste. Le procédé peut éliminer le risque d'interférence de sperme excessif et de contamination de cellule granulaire de la mère lors de l'utilisation de techniques de FIV et d'ICSI, et peut déterminer avec précision l'état de santé d'un embryon.
PCT/CN2019/129196 2018-12-29 2019-12-27 Procédé et produit pour détecter l'état de santé d'un embryon à l'aide d'une solution de culture de blastocyste WO2020135713A1 (fr)

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CN201980068400.7A CN113286892B (zh) 2018-12-29 2019-12-27 一种利用囊胚培养液检测胚胎健康状况的方法和产品
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EP19903766.4A EP3904529A1 (fr) 2018-12-29 2019-12-27 Procédé et produit pour détecter l'état de santé d'un embryon à l'aide d'une solution de culture de blastocyste

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CN111440857B (zh) * 2020-03-11 2023-03-21 阿吉安(福州)基因医学检验实验室有限公司 用于无创胚胎植入前遗传性检测的方法
CN112582022B (zh) * 2020-07-21 2021-11-23 序康医疗科技（苏州）有限公司 用于无创胚胎移植优先级评级的系统和方法
CN113862303A (zh) * 2020-08-24 2021-12-31 北京希诺谷生物科技有限公司 利用体细胞克隆制备克隆马胚胎的方法
CN114107182A (zh) * 2022-01-07 2022-03-01 徐维海 一种提高囊胚形成率的方法
CN114752552A (zh) * 2022-05-07 2022-07-15 序康医疗科技（苏州）有限公司 冷冻胚胎的复苏、培养、优选和单囊胚移植方法以及用于复苏冷冻胚胎的无创检测方法
CN115678964B (zh) * 2022-11-08 2023-07-14 广州女娲生命科技有限公司 基于胚胎培养液的植入前胚胎的无创筛选方法

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